Haemophilia is a sex-linked disease caused by a deficiency in certain circulating blood clotting factors. Haemophilia A is one such disease associated with a deficiency in coagulation Factor VIII. Haemophilia B is another such disease resulting from a deficiency in the levels of coagulation Factor IX. Haemophilia B is five to seven times less common than Haemophilia A, and is transmitted in humans as a chromosome X-linked recessive trait. Accordingly, occurrence of the disease is almost exclusively in males who receive the defective gene from their carrier mothers.
Although any individual having the defective genotype for Haemophilia B will be deficient in serum Factor IX, such individuals will vary greatly in the severity of the deficiency. Clinically, any individual having serum deficiency in Factor IX five to twenty-five percent (5-25%) of the levels typically observed in normal serum are classified as mild cases. Those individuals having deficiencies one to five percent (1-5%) of normal are considered moderate, and those having deficiencies less than one percent (1%) are considered severe cases. Approximately twenty to fifty percent (20-50%) of normal Factor IX levels is required for minimal hemostasis. Below this range there is a tendency to hemorrhage, with more severe bleeding being life threatening. It is therefore important clinically to be able to monitor accurately the levels of serum Factor IX in Haemophilia A patients, so as to timely implement appropriate therapy.
Other medical conditions exist which require accurate Factor IX determinations. Certain drug therapy, for example, warfarin treatment, is known to influence Factor IX levels. Also patients suffering from consumptive coagulopathies such as thrombosis or disseminated intravascular coagulation (DIC) may present anomalies in Factor IX levels which require careful clinical management. Successful treatment of these conditions similarly requires accurate determination of serum Factor IX levels. For a general review of the physiological and biochemical aspects of Factor IX deficiency diseases in relation to normal blood clotting see R. Coreman, Ed., Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 2e Ed., Lippincott, Pa. 1989.
In managing any of the aforementioned medical conditions, one mode of treatment involves administration of exogenous Factor IX obtained by fractionation of whole blood plasma, utilizing techniques well-known to those experienced in the art. Factor IX obtained by such techniques is ordinarily concentrated so as to be administered in a convenient-size dose. It is essential that the precise concentration of such therapeutic doses be measured, and the quantity of Factor IX be carefully monitored at each step of the purification process.
Accordingly, there are available in the prior art, methods for the quantitative determination of blood coagulation Factor IX. The most significant method, which has become the standard assay in this field, is known as the Activated Partial Thromboplastin Time (APTT). In this method, the percent of Factor IX present in a test sample is determined by the degree of correction obtained when the plasma is added to a Factor IX deficient plasma. The degree of correction is determined by activated partial thromboplastin time. Results are compared to the degree of correction obtained when dilutions of normal plasma are added to the Factor IX deficient reference plasma.
In the APTT assay, according to Matchett and Ingram, Partial Thromboplastin Time Test with Kaolin, J. Clin. Path., 18:465 (1965), a plasma sample is incubated in a buffered solution containing Kaolin to which is then added a suspension of inosithin. The clotting time is measured and the data is plotted against a standard curve.
The method of the prior art has been studied and improved many times and may be conducted in either one or two stages. However, all the variations in method have the disadvantage of high variability in results, particularly in low level determination of Factor IX. This lack of sensitivity and reproducibility occurs at the very levels of detection when accuracy is most crucial.
Another major disadvantage of the APTT method is that drugs such as heparin interfere with the assay leading to false or misleading results. Still further disadvantages include the requirement for large quantities of Factor IX deficient plasma, which is difficult and expensive to procure, and the difficulty in automating the procedures. Finally, the APTT and related methods are not specific for Factor IX, but are applicable to the assay of several blood clotting factors. All such assays rely upon the presumption that "normal" plasma will give a one hundred percent (100%) correction in thromboplastin time. This means that persons performing the assay are subjected to the inconvenience of constructing a standard curve each time the assay is run.